Sensitive color indicators for lipase determinations

ABSTRACT

IN LIPASE DETERMINATIONS OF BODY FLUIDS, E.G. SERUM, BY USING A FATTY OR OILY EMULSION SUBSTRATE, THE TITRATION OF LIBERATED FATTY ACID IS FACILITATED BY EMPLOYING ONE OR MORE INDICATORS OF THE GROUP M-CRESOL PURPLE, P-XYLENOLSULFONEPHTHALEIN, DIBROMOTHYMOLSULFONEPHTHALEIN, THYMOLSULFONEPHTHALEIN AND A-NAPHTHOLPHTHALEIN.

United States Patent Office Patented Sept. 5, 1972 3,689,364 SENSITIVE COLOR INDICATORS FOR LIPASE DETERMINATIONS Arnold Hiirtel, Roland Helger, and Hermann Lang,

Darmstadt, Germany, assignors to Merck Patent Gesellschaft mit beschrankter Haftung, Darmstadt, Germany No Drawing. Filed Sept. 15, 1969, Ser. No. 858,154 Claims priority, application Germany, Sept. 20, 1968, P 17 98 285.3 Int. Cl. G01n 31/14 US. Cl. 195-103.5 R 22 Claims ABSTRACT OF THE DISCLOSURE In lipase determinations of body fluids, e.g. serum, by using a fatty or oily emulsion substrate, the titration of liberated fatty acid is facilitated by employing one or more indicators of the group m-cresol purple, p-xylenolsulfonephthalein, dibromothymolsulfonephthalein, thymolsulfonephthalein and a-naphtholphthalein.

BACKKGROUND OF THE INVENTION This invention relates to a method of analyzing body fluids for the presence of lipases and to improved color indicator compositions for this purpose.

Lipases are enzymes which split fats and oils into glycerides and fatty acids. They are widely prevalent in the animal organism, with particularly high concentrations being found in the liver and in the secretion of the pancreas digesting the nutritive fats in the small intestine. When the pancreas is diseased, lipase enters the bloodstream. For this reason, the determination of lipase in the serum is an important diagnostic tool for the recognition of pancreatic diseases.

Heretofore, the lipase was generally determined by providing an excess of a lipase substrate and measuring the amount of fatty acid produced by the lipase. This measurement was accomplished by titration with an alkali, using an indicator, or by the extraction of copper salts. The specific indicators employed for the titrations were thymolphthalein and phenolphthalein, respectively (Deutsche Medizinische Wochenschrift [German Medical Weekly], vol. 90, p. 1170 (1965) and Analytical Biochemistry, vol. 6, p. 451 [1963]). As the substrate, an oil-in-water emulsion was generally employed.

The precise determination of lipase according to the aforementioned conventional processes is, however, beset with considerable difliculties, thereby discouraging widespread use of same in clinical laboratories. For example, the substrate cannot be readily handled and because of its turbidity it is impossible to conduct photometric measurements by transmitted light. Furthermore, it is extremely disadvantageous that the amount of acid to be determined is extraordinarily small; e.g. even when employing 1 ml. of serum, with a minutes incubation, only about 0.0005 millimol of acid is normally obtained, which corresponds to a consumption of only 0.005 ml. of a 0.1 N solution of sodium hydroxide during the titration. Therefore, it is necessary to employ very expensive automatic microtitration machines; otherwise the titration technique involves very long incubating periods, relatively large amounts of serum, and inaccurate results.

Because of these difliculties, it is sometimes possible in practice to employ an esterase determination instead, i.e., a soluble ester of lower chain length is employed as the substrate, which ester though well-defined, is hydrolyzed not only by the lipase but also by other esterases contained in the serum (cf. Aerztliches Labor [Medical Laboratory], vol. 13, 157 [1967]). The use of such a soluble ester is feasible in the lipase determination in enriched enzyme preparations, but not in the serum, since the lipase concentration in the serum is very slow (normal range up to 0.1 u/ml.), and the significant concentrations of several other esterases obscure the results. Accordingly, heretofore, for the determination of the socalled genuine lipase there was no choice but to employ the above-mentioned slow and expensive methods.

SUMMARY OF THE INVENTION A principal object of this invention is to provide an improved means for a lipase determination based on an oil/water emulsion substrate. Upon further study of the specification and appended claims, other objects and advantages of this invention will become apparent.

To attain these objects, there is provided a composition for the determination of lipase in body fluids, said composition comprising an aqueous fatty or oily emulsion and a pH color indicator, the improvement being that, as the color indicator, there is employed m-cresol purple and/ or p-xylenolsulfonephthalein and/ or dibromothymolsulfonephthalein and/or thymolsulfonephthalein (thymol blue) and/or a-naphtholphthalein.

Based on these indicators, the invention also is directed to reagent combinations and processes for using same, especially for the detection of abnormal amounts of lipase in serum.

DETAILED DISCUSSION OF THE INVENTION It is a particular advantage of this invention that only a very small quantity of the specimen to be examined is required (about 0.2 ml. in case of a normal determination, or 0.02 ml. for a microdetermination). Furthermore, the novel reagent combination and the novel process based thereon exhibit the advantage that-in contrast to the processes operating with conventional agents and with the microtitration device-the need for complicated apparatuses and specially trained personnel is eliminated. Still further, the novel process is considerably faster as compared to the conventional processes wherein the amount of acid liberated by the lipase must be determined by titration with the use of indicators. Moreover, a multiple pipetting required in the conventional processes, is avoided by the use of the novel reagent. Finally, a very important advantage of the invention is the resultant high specificity and precision of the lipase determination.

These excellent results, combined with the ease of handling of the novel reagent, could nowise be foreseen in view of the state of the art. Rather, since the substrate and the indicator are exposed to the eflect of the lipase in a protein-containing solution, a shift in the changeover range was to be expected for the color indicators to be employed in accordance with this invention. Furthermore, it is known that the changeover range of an indicator generally not only shifts in a protein-containing solution, but also, in most cases, broadens so that accurate pH readings are made difficult. Besides, such a broadening of the changeover range is likewise caused by the addition of emulsion stabilizers, e.g. gum arabic. Furthermore, unless an additive is entirely colorless, as is not the case for example in gum arabic, the color reading is again hindered. In view of these factors, the excellent precision obtainable by means of the novel reagent is indeed quite surprising.

The composition of the reagent of this invention Will be described in detail below:

Among the above-mentioned color indicators contained in the agent of this invention, m-cresol purple and pxylenolsulfonephthalein are preferred. The indicators exhibit an abrupt color change, for example, m-cresol purple from violet to yellow, and p-xylenolsulfonephthalein from blue to yellow. When preparing the reagent of the invention, the pH of the solution is suitably adjusted so that the color change occurs by the addition of a minimal amount of acid. For example when employing m-cresol purple, a brown-tinged purple shade is set, and when using p-xylenolsulfonephthalein, the solution is adjusted to a browntinged blue shade.

The adjustment to the desired pH is advantageously carried out by adding to the emulsion an alkaline solution of the indicator, for example a solution of m-cresol purple or p-Xylenolsulfonephthalein in a dilute, e.g. 0.1 N, sodium hydroxide solution, and thereafter adding an acid, for example dilute hydrochloric acid or another dilute mineral acid, until the desired color shade is obtained. Basically it is also possible to add the indicator in an aqueous acid, e.g. as a suspension or as a solution, and then set the desired color hue by the addition of an alkali, for example dilute solution of sodium hydroxide. However, in general, due to the more satisfactory solubility, the addition of the indicator in an alkaline solution is preferred.

In case the agent is stored for a longer period of time prior to the lipase determination, it is advisable, in order to prolong the shelf life, to add the indicator only shortly before use to the emulsion. In this case, the emulsion is at first prepared (without the indicator) with a preferably neutral or only weakly alkaline or weakly acidic pH, and separately therefrom, an alkaline indicator solution is produced. Both solutions show a very good shelf life even over extended periods of time. The indicator is employed in the ready-to-use emulsion in a concentration of generally about 0.001-1%, preferably 0.05-0.2%, based on the weight of the entire emulsion.

In most cases, it is preferred that the reagent of this invention contain only one of the above-disclosed compounds (m-cresol purple, p-xylenolsulfonephthalein, dibromothymolsulfonephthalein, thymolsulfonephthalein or a-naphtholphthalein). However, the indicator can also be comprised of a mixture of 2 or more of these compounds. In particular, it can be advantageous in some cases to employ, in addition to a main indicator, e.g. m-cresol purple or p-xylenol blue, another of said indicators in a low concentration, in order to adjust the hue of the reagent of this invention to that desired for the changeover range. Such as adjustment may be necessary, for example, when it is desired to shift the hue of the main indicator so as to have a better comparison with a printed color scale. For example, by adding a minor amount of ix-naphtholphthalein, which is blue in the alkaline range, to a compound employed as the main component of the color indicator, such as m-cresol purple or p-xylenosulfonephthalein, the color hue of the reagent in the alkaline range can be, in total, shifted to blue.

Such color shifts of the reagent of this invention can also be obtained by the addition of small amounts of other color indicators than those mentioned above, for example by the addition of diphenol purple (red shift, especially in the neutral range, when employing m-cresol purple as the main component of the color indicator) or in some cases also by the addition of small amounts of another color compound, such as carotene, by means of which a yellow hue is obtained. These compounds are added, for example, in a concentration of up to 0.05% (based on the emulsion).

As the lipase substrate, the reagent of this invention contains any natural or synthetic fatty acid ester, particularly fatty acid glycerol esters. In general, it is advantageous to employ triglycerides or optionally also diglycerides of fatty acids, such as palmitic, stearic, oleic, linoleic or lauric acid. Thus, natural oils and fats, for example coconut oil, are suitable as a lipase substrate for the agent of this invention. Particularly preferred is olive oil. It is advantageous to remove any free fatty acids from the oils and fats before the latter are used as the lipase substrate, for example by purifying the oil or fat by chromatography over a basic adsorbent, such as aluminum oxide. Optionally, the oils and fats can also be purified in a dilferent manner, or also hydrogenated. For example, hydrogenated colza oil is likewise suitable for use as the lipase substrate. The concentration range, generally used in the reagent of this invention, is l to 30, preferably 10 to 25 parts by volume of lipase substrate to 99 to 70, preferably 90 to 75 parts of water. Particularly advantageous in a range of 20 parts by volume of lipase substrate to parts by volume of water.

To increase the stability of the emulsion, the agent of the invention generally contains at least one emulsion stabilizer. For example, particularly suitable proved to be compounds having a protective colloid efiect, particularly gum arabic. If desired, polyvinylpyrrolidone can also be employed as the protective colloid. These emulsion stabilizers having a protective colloid effect, especially gum arabic, are present in the emulsion in a concentration of about 05-30%, preferably 10-25% by Weight based on the weight of emulsion. In addition to exhibiting a stabilizing effect, gum arabicum exerts an activating influence on the lipase.

Aside from protective colloids for stabilizing the emulsion, ordinary emulsifiers for oil/ water or fat/water emulsions can also be employed in the reagent of this invention, for example, polyethylene glycol fatty alcohol ethers, especially also polyoxyethylene derivatives of sorbitol anhydrides, such as polyoxyethylene sorbitan oleates, palmitates, stearates, for example polyoxyethylene sorbitan monopalmitate, monooleate, monostearate or trioleate or tristearate, polyethylene glycol al'kyl phenol ethers, sulfonic acid salts or sulfuric acid esters, for example isopropyl-naphthalenesulfonic acid salts or cetyl-, stearylsul-furic acid esters. The concentration of these emulsifiers ranges, for example, up to 20%, preferably up to 1% by Weight based on the weight of emulsion.

As a further additive for stabilizing the emulsion and simultaneously exerting an activating effect on the lipase to be measured in case of a pancreas lipase determination, it is possible to employ in the reagent of the invention salts of bile acids, for example, alkali cholates, alkali deoxycholates, alkali taurocholates, alkali glycocholates, and other bile acid salts. These salts are employed in a concentration of up to about 2%, particularly up to 0.5% by weight based on the weight of emulsion.

As a further suitable additive, the emulsion can contain substances, for purposes of stabilization, which prevent settling, for example the montmorillonite gelling agents, particularly alkaline earth or quaternary ammonium salts of montmorillonite fatty acids. It is possible, for example, to employ in the emulsion of this invention the extremely finely divided magnesium mont-morillonites ordinarily used as emulsion stabilizers for oil-in-water systems, having a density of 2.4 g./cm. and a moisture content of about 6%, or a dimethyl dioctadecyl-ammonium montmorillonite having a density of 1.80 g./cm. and a moisture content of less than 3%. These antisettling agents are employed in a concentration of up to about 3%, particularly up to 1%. The use of antisettling agents of the type above described in the field of the lipase determining is novel.

Furthermore, the reagent of this invention can contain emulsion stabilizers which increase the viscosity of emulsions at low temperatures. For this purpose, gel-forming, higher-molecular weight compounds are suitable, for example, which are not split by esterases. In particular, agar proved to be suitable as an additive for increasing viscosity and thus for stabilizing the emulsion. The application of agar in a reagent for the determination of lipase is novel.

Besides, the reagent of the present invention can also contain reducing compounds for preventing autoxidation and ensuing separation of the emulsion, preferred reducing agents being mercaptoethanol, cysteine, a-tocopherol acetate and/or ascorbyl palmitate in concentrations of up to about 1%, preferably up to 0.05% by Weight based on the emulsion. Tocopherol acetate and ascorbyl palmitate exhibit the advantage that they are soluble in fats and oils, respectively. The described reducing compounds are applied by the reagent of this invention in the field of the lipase determination for the first time.

These agents of the invention can also contain biostatic compounds (preservatives) for stabilizing the emulsion, which substances prevent microorganisms from attacking the substrate. Suitable in this connection are, for example, organomercury compounds such as p-chloromercuribenzoate, phenylmercuric acetate, ethylmercurithiobenzo-3,4- oxazole-l-carboxylic acid or the sodium salt thereof, ethylmercurithiosalicylates, ethylmercurithiophenolsulfonic acids, p-hydroxybenzoic acid esters, particularly the methyl or ethyl or isopropyl ester, hexamethylenetetramine, iodoacetates and/or azides. Preferably, sodium iodoacetate and sodium ethylmercurithiosalicylate are employed for this purpose. The preservatives are used preferably in a concentration of up to about 1%, particularly in a concentration of up to about 0.1% by Weight based on the weight of the emulsion.

In the reagent of this invention, at least one compound from the classes of substances set forth above is used as the emulsion-stabilizing additive with gum arabic being preferred. In a further preferred embodiment of the invention, preservatives are used in the reagent of this invention together with gum arabic. In general, those emulsion stabilizers are particularly suitable which are not affected by esterases, particularly by esterases other than lipase, in a weakly alkaline range.

When preparing the reagent of the invention, normally a solution of at least one emulsion stabilizer, e.g. gum arabic, in water is provided. In some cases, it is advisable to filter or centrifuge this solution before preparing the emulsion, in order to remove residues from the additives, for example from the gum arabic. Thereafter, the emulsion is formed by the addition of the substrate, e.g. olive oil, under vigorous stirring. The other additives employed for the agent, for example additional emulsion-stabilizing, especially preservatives, can be admixed to the mixture either prior to or after the addition of the substrate.

The indicator can suitably be added to the emulsion in the form of a solution. In this way, the emulsion is obtained in a form ready to use. It is also possible to store the indicator separately from the emulsion in the form of a solution and add it to the emulsion just prior the lipase determination. This embodiment of the agent of the invention is preferred when a long shelf life is required.

It is especially advantageous to fill the oil/water emulsion, in a quantity required for one lipase determination, into a suitable container, preferably into a plastic cuvette having a rectangular base. Optionally, the required indicator can also in this connection be made available separately from the emulsion in a special solution. In any case the quantity of the emulsion is metered into the cuvette so that it is suflicient for one lipase determination. For example, in this embodiment, 1 or 2 ml. of emulsion is filled into the cuvette. In the indicator solution, optionally separate from the emulsion, the concentration of the indicator is suitably adjusted so that an aliquot portion (which can be measured in a simple manner, for example one drop of the indicator solution) is added to the oil/ water emulsion made ready beforehand, for example in the plastic cuvette, for purposes of one determination. An oil/water emulsion containing theindicator can be stored for about 4 weeks, it being recommended to shake the emulsion before use.

If the oil/water emulsion with the indicator, according to the invention, is filled beforehand into a cuvette for one determination, it is advantageous to provide the contents of the cuvette with a film-forming coating. This coating act like a cover and prevents the contents of the cuvette, at low temperatures and with the cuvette being stored upside down from solidifying underneath the seal of the cuvette opening; otherwise such solids would be forced out of the opening by a minor excess pressure produced in the cuvette. This cover, according to the invention, is advantageously formed as a layer on the emulsion by coating the latter with a liquid having little or no miscibility with water, i.e. substantially immiscible, which liquid is of a lower specific gravity than the emulsion and solidifies some time after being applied to the emulsion. Preferred materials for the coating are solutions of natural, synthetic or semisynthetic polymers, e.g. a polyethylene derivative, such as polystyrene, polyvinyl acetate, polyvinyl chloride or polymethacrylates, or a cellulose derivative, for example a nitrated cellulose, such as collodion, or cellulose acetate, or epoxy lacquers or two-component urethane lacquers or urethane lacquers hardening under the effect of moisture, in readily evaporating organic solvents or solvent mixtures. For example, it is possible to employ for this purpose ethers, e.g. diethyl ether; lower alcohols, such as ethanol; lower esters, such as ethyl acetate; or low-boiling hydrocarbons which, if desired, can also be substituted, for instance, by chlorine, such as diehloromethane, or ketones, such as, for example, acetone or cyclohexanone.

Optionally, a plasticizer, such as camphor, for example, can also be added. The film-forming solid substances are preferably employed in each case in such a concentration that the thus-produced coating above the emulsion attains a thickness of up to 3 mm., preferably about 0.5 mm.

After the solvent has evaporated, a thin membrane forms on the emulsion, which membrane adhers firmly to the walls of the cuvette. This membrane prevents the emulsion from running to the opening of the cuvette when the cuvette is stored with the opening on the bottom. A membrane which proved to be advantageous was obtained by coating the emulsion in a cuvette with about 0.05 ml. of a 2% collodion solution. The cuvettes or vials preferred for use in storing the reagent of this invention is shown and described for example in US. Pats. 2,258,073 and 3,190,731.

To use the reagent of this invention, it is combined with the lipase-containing sample to be examined, and the color change of the indicator incurred during the incubating time is evaluated. For this purpose, a small metered volume, preferably 0.1 ml., of the body fluid to be tested, for example serum or plasma, is introduced into a metered volume, for example 1 ml., of the ready-touse emulsion. Thereafter, the mixture is thoroughly intermixed, as usual, and the color change is measured. The color change can be measured by comparing the hue of the mixture with the corresponding hue on a calibrated color scale or on a calibrated color strip. For this purpose, for example, there is recorded the calibration value read off from the color scale or the color strip for the hue present directly after addition of the sample to the emulsion. Thereafter, the charge is allowed to stand for a specific incubation period, for example 1 hour, preferably at a standard temperature, e.g. room temperature or 37 C., and then the value associated with the resultant produced color hue is read off from the calibrated color scale or the calibrated color strip. The difference yields the lipase content in the units on which the color scale or color strip calibration is based.

A calibrated color scale or a calibrated color strip used during the measurement can be produced as described in French Pat. 1,516,492. For the calibration of the scale or the strip, the time interval for the incubation, for example 1 hour is used as the basis.

In another embodiment of the process of this invention, the color change is determined by a comparison of the examined specimens with one another after the incubation period of, for example, one hour. In this method, the hue of specimens having a normal lipase content-- which is almost unchanged after the incubation period is employed as the standard. This maner of conducting the lipase determination according to the invention is especially suitable for series analyses, since in most cases over of the blood serums examined in the clinics exhibit a normal lipase content and thus can serve as the standard. The few specimens showing a pathological increase in the lipase content can then be recognized by a hue which differs from the nearly unchanged color of the plurality of the examined specimens after the incubation period. When employing the indicators m-cresol purple or p-xylenolsulfone-phthalein, the pathological specimens exhibit a light-brown to light-yellow color. If a more accurate determination is required, the specimens recognized as being pathological can be measured once more, this time more accurately, by a comparison with the calibrated color strip or the calibrated color scale.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

EXAMPLE 1 In a high speed mixer, 30 mg. of ethylmercurithiosalicylate and 90 mg. of gum arabic are added to 480 ml. of water. Thereafter, the mixture is adjusted to a pH of 10 by the addition of 2 N aqueous solution of sodium hydroxide. Thereupon, the mixture is subjected to centrifuging and the residue is filtered through a cotton swab in order to remove impurities. The filtrate is then mixed in a high speed mixer with 120 ml. of olive oil (purified over basic aluminum oxide) optionally with the addition of an emulsifier, for example a polyoxyethylene sorbitan oleate, palmitate or stearate. The reaction mixture is mixed for an additional 5 minutes and then blended with 600 mg. of m-cresol purple dissolved in 60 ml. of 0.1 N sodium hydroxide solution. By the addition of a dilute acid, for example dilute hydrochloric acid, the pH of the mixture is adjusted to 8.8 (color: brown-tinged violet).

In a modification of the above-mentioned procedure, the following indicators are employed in place of 600 mg. of

m-cresol purple:

Mg- Dibromosnlfonephthalein (adjustment of pH to 8.8,

color blue) 600 Thymolsulfonephthalein (adjustment to a pH of 8.8,

color blue with greenish tinge) 500 a-Naphtholphthalein (adjustment to a pH of 9 color light-blue) 600 The thus-produced emulsion is either employed directly for the lipase determination or filled in 1 ml. batches into scalable plastic cuvettes. Suitably, the emulsion, in each plastic cuvette, is provided with a layer of about 0.05 ml. of a 2% collodion solution. After the solvent has evaporated, a thin membrane is formed on the emulsion. Thereafter, the cuvettes are sealed, the contents of each plastic cuvete being suitable for one lipase determination.

EXAMPLE 2 An emulsion is prepared analogously to the technique described in Example 1, except that no indicator is added, and the pH is adjusted to 7.0. Separately from the emulsion, which is filled into cuvettes of 2 ml, are prepared: (1) a 1% solution of m-cresol purple in a 0.055 N sodium hydroxide solution; (2) a 0.02 N solution of sodium hydroxide; and (3) a 0.02 N solution of hydrochloride acid. The 0.02 N sodium hydroxide or hydrochloric acid solution can be employed for regulating the pH of the emulsion altered after an extended period of storage.

EXAMPLE 3 Analogously to Example 1, a ready-to-use emulsion is prepared from the following components:

The color of the emulsion is adjusted to a brown-tinged blue b the addition of dilute solution of sodium hydroxide or hydrochloric acid.

In a modified embodiment, 8 mg. of carotene or diphenol purple is added to the above-described mixture.

The resultant composition can be employed for the lipase determination as set forth in Example A.

EXAMPLE 4 Analogously to Example 3, an emulsion is prepared but without an indicator, the pH being adjusted to 7.0. Separate solutions are then prepared of: a 1% solution of p-xylenol blue in 0.055 N sodium hydroxide solution;- a 0.02 N solution of sodium hydroxide; and a 0.02 N hydrochloric acid solution.

EXAMPLE 5 23 g. of gum arabic 4 mg. of sodium ethylmercurithiosalicylate and 25 mg. of sodium azide are dissolved in 120 ml. of water.

This solution is introduced into a homogenizer, and during the operation of same, there is added a solution of 5 mg. of a-tocopherol acetate and 5 mg. of ascorbyl palmitate in 30 ml. of olive oil.

In a modified embodiment, 45 mg. of sodium deoxycholate or sodium taurocholate is added to the abovedescribed emulsion.

An indicator solution, prepared by dissolving 1 g. of cresol purple, optionally together with mg. of

methylene blue, in 100 ml. of 0.05 N solution of sodium hydroxide,

is mixed in a volume ratio of 1:10 with the above-described emulsion, so that, for example, 2 ml. of emulsion is mixed with 0.2 ml. of indicator solution.

The resultant lipase test reagent is employed in the same manner as the compositions of Example 14.

EXAMPLE 6 3 g. of gum arabic 20 ml. of sodium iodoacetate 1 g. of magnesium montmorillonite or dimethyl diocta-' decylammonium montrnorillonite and 0.5 g. of agar are dissolved or made into a slurr with 100 m1. of water.

The solution is homogenized in a mixer with 25 ml. of purified triolein.

One hundred parts by volume of the thus-prepared emulsion are mixed with one part by volume of a solution of 1 g. of cresol purple in 100 ml. of 0.05 N sodium hydroxide solution.

The resultant test reagent is employed analogously to the compositions of Examples 1-4.

The following examples describe in greater detail how the process of this invention is conducted.

EXAMPLE A 0.1 ml. of serum is introduced into the cuvette filled with 1 ml. of the emulsion prepared according to Example 1. The mixture is shaken, and the resultant color hue is compared with a calibrated color scale. The number associated with the color hue on the scale is read off, for example 150. Then, the specimen is allowed to stand for 1 hour at room temperature and, by comparison of the thusproduced color hue with the same color hue on the calibrated color scale, the number 350 is obtained. The lipase content of the measured specimen is, in that case, 200 mu/ml.

The same result is achieved .when employing the readyto-use emulsion of Example 3.

In a repetition of the above-described measurement procedures, the specimens, after determining the initial color hue, are allowed to stand for 20 minutes at 37 C. The thus-determined lipase content is the same as obtained in the above tests.

EXAMPLE B In a series test, 0.1 ml. of serum is mixed with 1 ml. of the emulsions produced in accordance with Examples 1 and 3, and then allowed to stand for 1 hour. In most of the specimens, the brownish-violet or brownish-blue color hue is hardly changed with respect to the initial color hue. The serums having a pathologically increased lipase content exhibit a brown to yellow or a dirty-green to yellow color. The exact lipase content in these pathological serums can then be determined exactly in accordance with Example .A.

EXAMPLE C 0.2 ml. of the indicator solution prepared according to Example 2 is added to 2 ml. of the emulsion of Example 2. Thereafter, the components are mixed, the pH indicator normally showing the desired color (brown tinged violet). When the pH' of the emulsion, for example due to extended storage periods, no longer exhibits the desired value, several drops of 0.02 N solution of sodium hydroxide or hydrochloric acid are added until the desired brown-tinged violet color hue is obtained.

Analogously, a ready-to-use emulsion is prepared with the indicator of Example 4. The correction of the pH of an emulsion stored for a longer period of time, necessary in some cases, is effected by the addition of the 0.02 N solution of sodium hydroxide or hydrochloric acid until a blue color hue is attained.

Serum 0.2 ml. is added to each of the ready-to-use emulsions, the mixture is shaken, and the lipase content is measured as described in Examples A and B.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

We claim:

1. A reagent combination suitable for direct colorimetric determination of serum lipase activity, comprising:

(a) an aqueous emulsion of a fatty acid glycerol ester;

(b) up to 20% by weight of an oil-in-water emulsifier;

() about 0.5 to 30% by weight of an emulsion stabilizer having a protective colloid effect; and

(d) about 0.001 to 1% by weight of a colorimetric indicator selected from the group consisting of mcresol purple and p-xylenolsulfonephthalein.

2. A reagent combination according to claim 1 further including up to 2% by weight of a bile acid salt.

3. A reagent combination according to claim 1 further including a biostatic preservative.

4. A reagent combination according to claim 1 wherein said emulsion stabilizer contains gum arabic.

5. A reagent combination according to claim 1 wherein said emulsion stabilizer increases the viscosityof said emulsion at low temperatures.

6. A reagent combination according to claim 5 wherein said emulsion stabilizer contains agar.

7. A reagent combination according to claim 1 further including up to 3% by weight of an anti-settling agent.

8. A reagent combination according to claim 7 wherein said anti-settling agent is an alkaline earth salt or quaternary ammonium salt of a montmorillonite fatty acid.

9. A reagent composition according to claim 1 further including up to 1% by weight of a reducing agent selected from the group consisting of mercaptoethanol, cysteine, alphatocopherol acetate, ascorbyl palmitate, and mixtures thereof.

'10. A reagent composition according to claim 9 wherein said reducing agent is alpha-tocopherol acetate or ascorbyl palmitate.

11. A reagent composition according to claim 1 further including up to 0.05% by weight of a second color indicator to shift the color hue of said colorimetric indicator.

12. A reagent composition according to claim 11 wherein said second color indicator is alpha-naphtholphthalein, diphenol purple, or carotene.

13. A reagent composition according to claim 1 wherein the color thereof is adjusted to a brown-tinged shade, whereby a color change occurs upon the addition of a minimal amount of acid.

14. A reagent combination according to claim 1 further comprising a cuvette for containing said reagent combination and further provided with an insoluble film of a substantially water-immiscible film on the open surface of said emulsion, said film adhering to the sides of said cuvette for preventing movement of said emulsion to the top of said cuvette when said cuvette is stored upside down.

15. A method for the determination of serum lipase activity which comprises admixing a reagent composition according to claim 1 and a serum sample; incubating the reagent-serum mixture; and determining the lipase activity in correspondence with the resultant color of the incubated mixture.

16. A method according to claim 15 wherein said reagent composition includes up to 2% by weight of a bile acid salt.

17. A method according to claim 15 wherein said reagent composition contains gum arabic.

18. A method according to claim 15 wherein said reagent composition contains agar.

19. A method according to claim 15 wherein said reagent composition includes up to 0.05% by weight of a second color indicator to shift the color hue of said colorimetric indicator.

20. A'method according to claim 15 wherein the color of said reagent composition is adjusted prior to the ad mixture thereof to a brown-tinged shade, whereby a color change occurs upon the subsequent addition of a minimal amount of acid.

21. A method according to claim 15 wherein said change in color is evaluated spectrophotometrically.

22. A method according to claim 21 wherein said reagent composition and said serum sample are admixed in a cuvette in which the resultant color change is evaluated.

(References on following page) 1 1 12 References Cited The Merck Index, 8th"ed., p 691 (1968). I UNITED STATES PATENTS (llgggtgon: Hydrogen Ions, vol; I, 4th ed., pp. 408-409 Stevens Bound volumes H kh Ch i l Di i a y, "P. OTHER REFERENCES 5 A. LOUIS MONACELL, Primary Examiner Amador et 2.1.: Methods of Biochem. Analysis, Ed, l Gfick vol. 13 p 310611 (1965). M. D. HENSLEY, Asslstant Exammer Tietz et al.: Am. J. Clin. Path., 31:148t (1959). Us. CL

Massion et al.: Am. J. Clin. Path.," 48(3):307t (1967). 10

Emma STATES PATENT @FIFIIQE (EERTEFEQAEE Q1? CQRREQ'HQN Patent No. 3,689,364 Dated September 5, 1972 I'nventofls) ARNOLD HARTEL ET AL Flt is certified that error appears in the above-identified patent i and that said Letters Patent are hereby corrected as' shown below:

IN'TH-E HEADING, under 'f'claims Priority", insert and application Germany, August 27, 1969, P 19 43 454.9

Signed and sealed this 10th day of July 1973'.

(SEAL) Attest:

EDWARD M.FLETCHER,J R.- Rene Tegtmeyer Attesting Officer Acting Commissioner of Patents DRM PO-1050 (10-69) USCOMM-DC 6O376-P69- it U.S. GOVERNMENT PRINTING OFFICE: 1969 O-366-334, 

